Rapamycin conjugates

ABSTRACT

Provided are rapamycin conjugates which are useful as immunogenic molecules for the generation of antibodies specific for rapamycin, for measuring levels of rapamycin or derivatives thereof; for isolating rapamycin binding proteins; and detecting antibodies specific for rapamycin or derivatives thereof. This invention also provides a rapamycin specific monoclonal antibody.

This application is a Divisional of U.S. patent application Ser. No.09/576,951, filed May 24, 2000; which in turn is a Continuation-in-partof U.S. patent application Ser. No. 08/424,983, filed Apr. 19, 1995 (nowabandoned); which in turn is a Continuation of U.S. patent applicationSer. No. 08/224,205, filed Apr. 14, 1994 (now abandoned); which in turnis a Continuation-In-Part of U.S. patent application Ser. No.08/053,030, filed Apr. 23, 1993 (now abandoned). The disclosure of eachof which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to derivatives of rapamycin which are useful asimmunogenic molecules for the generation of antibodies specific forrapamycin, for measuring levels of rapamycin or derivatives thereof; forisolating rapamycin binding proteins; and detecting antibodies specificfor rapamycin or derivatives thereof.

Rapamycin is a macrocyclic triene antibiotic produced by Streptomyceshygroscopicus, which was found to have antifungal activity, particularlyagainst Candida albicans, both in vitro and in vivo [C. Vezina et al.,J. Antibiot. 28, 721 (1975); S. N. Sehgal et al., J. Antibiol. 28, 727(1975); H. A. Baker et al., J. Antibiot. 31, 539 (1978); U.S. Pat. No.3,929,992; and U.S. Pat. No. 3,993,749].

Rapamycin alone (U.S. Pat. No. 4,885,171) or in combination withpicibanil (U.S. Pat. No. 4,401,653) has been shown to have antitumoractivity. R. Martel et al. [Can. J. Physiol. Pharmacol. 55, 48 (1977)]disclosed that rapamycin is effective in the experimental allergicencephalomyelitis model, a model for multiple sclerosis; in the adjuvantarthritis model, a model for rheumatoid arthritis; and effectivelyinhibited the formation of IgE-like antibodies.

The immunosuppressive effects of rapamycin have been disclosed in FASEB3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclic molecules,also have been shown to be effective as immunosuppressive agents,therefore useful in preventing transplant rejection [FASEB 3, 3411(1989); FASEB 3, 5256 (1989); R. Y. Calne et al., Lancet 1183 (1978);and U.S. Pat. No. 5,100,899].

Rapamycin has also been shown to be useful in preventing or treatingsystemic lupus erythematosus [U.S. Pat. No. 5,078,999], pulmonaryinflammation [U.S. Pat. No. 5,080,899], insulin dependent diabetesmellitus [Fifth Int Conf. Inflamm. Res. Assoc. 121 (Abstract), (1990)],adult T-cell leukemia/lymphoma [European Patent Application 525,960 A1],and smooth muscle cell proliferation and intimal thickening followingvascular injury [Morris, R. J. Heart Lung Transplant 11 (pt 2): 197(1992)].

Mono- and diacylated derivatives of rapamycin (esterified at the 28 and43 positions) have been shown to be useful as antifungal agents (U.S.Pat. No. 4,316,885) and used to make water soluble prodrugs of rapamycin(U.S. Pat. No. 4,650,803). Recently, the numbering convention forrapamycin has been changed; therefore according to Chemical Abstractsnomenclature, the esters described above would be at the 31- and42-positions. U.S. Pat. No. 5,100,883 discloses fluorinated esters ofrapamycin. U.S. Pat. No. 5,118,677 discloses amide esters of rapamycin.U.S. Pat. No. 5,118,678 discloses carbamates of rapamycin. U.S. Pat. No.5,130,307 discloses aminoesters of rapamycin. U.S. Pat. No. 5,177,203discloses sulfonates and sulfamates of rapamycin. U.S. Pat. No.5,194,447 discloses sulfonylcarbamates of rapamycin. PCT Publication WO92/05179 discloses carboxylic acid esters of rapamycin.

Yatscoff has reported that rapamycin levels can be quantitated usingHPLC method with a sensitivity of 1 ng/ml [Ther. Drug Monitoring 14: 138(1992)] This method is time consuming and each sample must be assayedindividually.

Immunoassays have been developed for numerous proteins as well asvarious drugs including cyclosporin A [Morris, R. G., Ther. DrugMonitoring 14: 226-(1992)], and FK506 [Tamura, Transplant Proc. 19: 23(1987); Cadoff, Transplant Proc. 22: 50 (1990)]. Numerous types ofimmunoassays, that have been developed to measure proteins or compounds,have been based on competitive inhibition, dual antibodies,receptor-antibody interactions, antigen capture, dipstick, antibody orreceptor trapping, or on affinity chromatography. Affinity columns withrapamycin have been reported in which a rapamycin analog was covalentlyattached to a matrix [Fretz J. Am. Chem. Soc. 113: 1409 (1991)]. Thesecolumns have been used to isolate rapamycin binding proteins.

DESCRIPTION OF THE INVENTION

This invention provides a rapamycin conjugate of formula I, having thestructure

-   wherein R¹ and R² are each, independently, hydrogen or    —(R³-L-R⁴)_(a)—;-   L is a linking group;-   R³ is selected from the group consisting of carbonyl, —S(O)—,    —S(O)₂, —P(O)₂—, —P(O)(CH₃)—, —C(S)—, and —CH₂C(O)—;-   R⁴ is a selected from the group consisting of carbonyl, —NH—, —S—,    —CH₂—, and —O—;-   a=1-5;-   x=0-1;-   y=0-1;-   z is from about 1 to about 120;-   and Carrier is immunogenic carrier material, detector carrier    material, or a solid matrix, or a salt thereof with the proviso that    R¹ and R² are both not hydrogen; and further provided that when a is    greater than 1, each L group can be the same or different; and still    further provided that x is 0 if R¹ is hydrogen and y is 0 if R² is    hydrogen, and if x and y are both 1, the Carrier moiety is the same    in both cases.

The linking group, L, is any moiety that contains the group R³ on oneend and R⁴ on other end therefore enabling the linking group to beconnected to the 42- and/or 31-hydroxyl groups of rapamycin on one endand connected to another linking group or the immunogenic carriermaterial, detector material, or matrix on the other end. When a isgreater than 1, each L group can be the same or different. In suchcases, the first L group is designated as L¹, the second L groupdesignated as L² and so on. The rapamycin conjugates of the presentinvention may be prepared in such ways as to encompass a wide range oflinking groups (L) and terminal functional groups R⁴. For example, L maybe linear or branched alkylenes comprising from 1 to as many as 15, moreusually 10 or less, and normally less than 6 carbon atoms (i.e.,methylene, ethylene, n-propylene, iso-propylene, n-butylene, and soforth). In addition, such alkylenes can contain other substituent groupssuch as cyano, amino (including substituted amino), acylamino, halogen,thiol, hydroxyl, carbonyl groups, carboxyl (including substitutedcarboxyls such as esters, amides, and substituted amides). The linkinggroup L can also contain or consist of substituted or unsubstitutedaryl, aralkyl, or heteroaryl groups (e.g., phenylene, phenethylene, andso forth). Additionally, such linkages can contain one or moreheteroatoms selected from nitrogen, sulfur and oxygen in the form ofether, ester, amido, amino, thio ether, amidino, sulfone, or sulfoxide.Also, such linkages can include unsaturated groupings such as olefinicor acetylenic bonds, disulfide, imino, or oximino groups. Preferably Lwill be a chain, usually aliphatic comprising between 1 and about 20atoms, more usually between 1 and 10, excluding hydrogen, of whichbetween 0 and 5 are heteroatoms preferably selected from nitrogen,oxygen, and sulfur. Therefore, the choice of linking group L is notcritical to the present invention and may be selected by one of ordinaryskill taking normal precautions to assure that stable compounds areproduced.

A preferred embodiment of this invention provides a conjugate of formulaII, having the structure

-   R¹ and R² are each, independently, hydrogen or —R³-L-R⁴—;-   L is -A-(CR⁵R⁶)_(b)[B—(CR⁷R⁸)_(d)]_(e)—-   A is —CH₂— or —NR⁹—;-   B is —O—, —NR⁹—, —S—, —S(O)—, or —S(O)₂—;-   R³ is selected from the group consisting of carbonyl, —S(O)—,    —S(O)₂, —P(O)₂—, —P(O)(CH₃)—, —C(S)—, and —CH₂C(O)—;-   R⁴ is selected from the group consisting of carbonyl, —NH—, —S—,    —CH₂—, and —O—;-   R⁵, R⁶, R⁷, and R⁸ are each, independently, hydrogen, alkyl of 1-6    carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon    atoms, halo, hydroxy, trifluoromethyl, arylalkyl of 7-10 carbon    atoms, aminoalkyl of 1-6 carbon atoms, hydroxyalkyl of 1-4 carbon    atoms, alkoxy of 1-6 carbon atoms, carbalkoxy of 2-7 carbon atoms,    cyano, amino, —CO₂H, or phenyl which is optionally mono-, di-, or    tri-substituted with a substituent selected from alkyl of 1-6 carbon    atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro,    carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, or —CO₂H;-   R⁹ is hydrogen, alkyl of 1-6 carbon atoms, or aralkyl of 7-10 carbon    atoms;-   b=0-10;-   d=0-10;-   e=0-2;-   x=0-1;-   y=0-1;-   z is from about 1 to about 120;-   and Carrier is immunogenic carrier material, detector carrier    material, or a solid matrix, or a salt thereof with the proviso that    R¹ and R² are both not hydrogen; and further provided that when b is    greater than 1, each of the CR⁵R⁶ groups can be the same or    different, and when d is greater than 1, each of the CR⁷R⁸ groups    can be the same or different; and still further provided that x is 0    if R¹ is hydrogen and y is 0 if R² is hydrogen, and if x and y are    both 1, the Carrier moiety is the same in both cases.

A second preferred embodiment of this invention provides a conjugate offormula III, having the structure

-   R¹ and R² are each, independently, hydrogen or    —(R³-L¹-R⁴)_(f)—(R¹⁰-L²-R¹¹)_(g)-carrier;-   L¹ is —(CH₂)_(h)—CHR¹²—(CH₂)_(j)—;-   L² is —(CH₂)_(k)-D-(CH₂)_(m)-E-;-   D is —CH₂—, —S—S—, or

-   E is —CH₂— or

-   R³ and R¹⁰ are each, independently, selected from the group    consisting of carbonyl, —S(O)—, —S(O)₂, —P(O)₂—, —P(O)(CH₃)—,    —C(S)—, and —CH₂C(O)—;-   R⁴ and R¹¹ are each, independently, selected from the group    consisting of carbonyl, —NH—, —S—, —CH₂—, and —O—;-   R¹² is hydrogen, alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon    atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,    —(CH₂)_(n)CO₂R¹³, —(CH₂)_(p)NR¹⁴R¹⁵, carbamylalkyl of 2-3 carbon    atoms, aminoalkyl of 1-4 carbon atoms, hydroxyalkyl of 1-4 carbon    atoms, guanylalkyl of 2-4 carbon atoms, mercaptoalkyl of 1-4 carbon    atoms, alkylthioalkyl of 2-6 carbon atoms, indolylmethyl,    hydroxyphenylmethyl, imidazoylmethyl, halo, trifluoromethyl, or    phenyl which is optionally mono-, di-, or tri-substituted with a    substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6    carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon    atoms, trifluoromethyl, amino, or —CO₂H;-   R¹⁴, and R¹⁵ are each, independently, hydrogen, alkyl of 1-6 carbon    atoms, or arylalkyl of 7-10 carbon atoms;-   R¹³ is hydrogen, alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon    atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or    phenyl which is optionally mono-, di-, or tri-substituted with a    substituent selected from alkyl of 1-6 carbon atoms, alkoxy of 1-6    carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon    atoms, trifluoromethyl, amino, or —CO₂H;-   f=0-3;-   g=0-1;-   j=0-10;-   k=0-10;-   m=0-10;-   n=0-6;-   p=0-6;-   x=0-1;-   y=0-1;-   z is from about 1 to about 120;-   and Carrier is immunogenic carrier material, detector carrier    material, or a solid matrix, or a salt thereof with the proviso that    R¹ and R² are both not hydrogen; and further provided that f and g    are both not 0 and when f is greater than 1, each of the    —(R³-L¹-R⁴)— moieties can be the same or different; and still    further provided that x is 0 if R¹ is hydrogen and y is 0 if R² is    hydrogen, and if x and y are both 1, the Carrier moiety is the same    in both cases.

This invention also provides a conjugate of formula IV, having thestructure

-   wherein R¹ is —OCH₂(CH₂)_(q)R⁴—;-   R⁴ is selected from the group consisting of carbonyl, —NH—, —S—,    —CH₂—, and —O—;-   q=0-6;-   z is from about 1 to about 120;-   and Carrier is immunogenic carrier material, detector carrier    material, or a solid matrix, or a salt thereof.

The immunogenic carrier material can be selected from any of thoseconventionally known. In most cases, the carrier win be a protein orpolypeptide, although other materials such as carbohydrates,polysaccharides, lipopolysaccharides, nucleic acids and the like ofsufficient size and immunogenicity can likewise be used. For the mostpart, immunogenic proteins and polypeptides will have molecular weightsbetween 5,000 and 10,000,000, preferably greater than 15,000 and moreusually greater than 40,000. Generally, proteins taken from one animalspecies will be immunogenic when introduced into the blood stream ofanother species. Particularly useful proteins are those such asalbumins, globulins, enzymes, hemocyanins, glutelins or proteins havingsignificant non-proteinaceous constituents, e.g., glycoproteins, and thelike. Further reference for the state-of-the-art concerning conventionalimmunogenic carrier materials and techniques for coupling haptensthereto may be had to the following: Parker, Radioimmunoassay ofBiologically Active Compounds, Prentice-Hall (Englewood Cliffs, N.J.,USA, 1976), Butler, J. Immunol. Meth. 7:1-24 (1975) and Pharmacol. Rev.29(2):103-163 (1978); Weinryb and Shroff, Drug Metab. Rev. 10:P271-283(1975); Broughton and Strong, Clin. Chem. 22:726-732 (1976); andPlayfair et al., Br. Med. Bull. 30:24-31 (1974). Preferred immunogeniccarrier materials for use in the present invention are ovalbumin andkeyhole limpet hemocyanin. Particularly preferred for use in the presentinvention is ovalbumin. The detector carrier material can be arapamycin-linking moiety conjugated to an enzyme such as horseradishperoxidase, alkaline phosphatase, luciferase, a fluorescent moiety suchas fluorescein, Texas Red, or rhodamine, a chemiluminescent moiety, andthe like. The solid matrix carrier material can be resin beads, an ELISAplate, glass beads as commonly used in a radioimmunoassay, plasticbeads, solid matrix material typically used in a dipstick-type assay.When rapamycin is conjugated to a solid matrix, the resulting conjugatecan be used in a dipstick assay, as described in this disclosure, forthe affinity purification of antibodies, or for isolating rapamycinbinding proteins.

It should be noted that as used in the formulae above describing thespecific rapamycin conjugates, z represents the number of rapamycinconjugated to the carrier material. The value z is sometimes referred toas the epitopic density of the immunogen, detector, or solid matrix andin the usual situation will be on the average from about 1 to about 120and more typically from 1 to 50. The densities, however, may varygreatly depending on the particular carrier material used.

When any of the compounds of this invention contain an aryl or arylalkylmoiety, it is preferred that the aryl portion is a phenyl, naphthyl,pyridyl, quinolyl, isoquinolyl, quinoxalyl, thienyl, thionaphthyl,furyl, benzofuryl, benzodioxyl, benzoxazolyl, benzoisoxazolyl, orbenzodioxolyl group that may be optionally mono-, di-, or tri-substituted with a group selected from alkyl of 1-6 carbon atoms,arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo,nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino,dialkylamino of 1-6 carbon atoms per alkyl group, alkylthio of 1-6carbon atoms, —SO₃H, —PO₃H, and —CO₂H. It is more preferred that thearyl moiety is a phenyl group that is optionally mono-, di-, ortri-substituted with a group selected from alkyl of 1-6 carbon atoms,arylalkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon atoms, cyano, halo,nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino,dialkylamino of 1-6 carbon atoms per alkyl group, alkylthio of 1-6carbon atoms, —SO₃H, —PO₃H, and —CO₂H.

The salts are those derived from such inorganic cations such as sodium,potassium, and the like; organic bases such as: mono-, di-, and trialkylamines of 1-6 carbon atoms, per alkyl group and mono-, di-, andtrihydroxyalkyl amines of 1-6 carbon atoms per alkyl group, and thelike; and organic and inorganic acids as: acetic, lactic, citric,tartaric, succinic, maleic, malonic, gluconic, hydrochloric,hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, andsimilarly known acceptable acids.

The compounds of this invention can be prepared by reacting the 42-and/or 31-hydroxyl groups of rapamycin with a suitable electrophilicreagent that will serve as the linker moiety. The following patentsexemplify the preparation of the 42- and/or 31-derivatives of rapamycinthat can be used as linking groups for the preparation of the compoundsof this invention. The preparation of fluorinated esters of rapamycin isdescribed in U.S. Pat. No. 5,100,883. The preparation of amide esters isdisclosed in U.S. Pat. No. 5,118,677. The preparation of carbamates ofrapamycin is disclosed in U.S. Pat. No. 5,118,678. The preparation ofaminoesters of rapamycin is described in U.S. Pat. No. 5,130,307. Thepreparation of sulfonates and sulfamates of rapamycin are described inU.S. Pat. No. 5,177,203. The preparation of sulfonylcarbamates ofrapamycin are described in U.S. Pat. No. 5,194,447. The disclosures ofthe above cited U.S. patents are hereby incorporated by reference. Fromthese patents, it can be seen that reactive electrophiles such asisocyanates, used in the preparation of carbamates, or sulfonylchlorides, used in the preparation of sulfonates, can be reacted withthe hydroxyl groups of rapamycin without the need for an activatingagent. For the esterification of the rapamycin hydroxyl groups with acarboxylic acid, activation is usually required through the use of acoupling reagent such as DCC, or a water soluble analog thereof, such asdimethylaminopropyl)-3-ethyl carbodiimide (DAEC). Representativeexamples of the preparation of rapamycin-linking group moieties areprovided as examples below. The preparation of ether derivatives ofrapamycin can be accomplished using the methodology disclosed in Example18.

For the compounds of this invention in which the linker group isattached to the 42- or the 31,42-hydroxyls, the electrophile (oractivated electrophile) is reacted with. rapamycin to typically providea mixture of the 42- and 31,42-derivatized rapamycin that can beseparated by chromatography. For the compounds of this invention inwhich the linker group is attached to the 31-hydroxyl of rapamycin, the42-hydroxyl group must be protected with a suitable protecting group,such as with a tert-butyldimethyl silyl group. The 31-hydroxyl can thenbe reacted with a suitable electrophile to provide the derivatizedrapamycin, followed by deprotection of the 42-hydroxyl group. Thepreparation of 42-O-silyl ethers of rapamycin and subsequentdeprotection is described in U.S. Pat. No. 5,120,842, which is herebyincorporated by reference. Preparation of compounds containing differentlinkers at the 31- and 42-positions can be accomplished by firstpreparing the 42-derivatized compound and then using a different linkerto derivatize the 31-position. The preparation of the 27-oxime linkinggroups can be accomplished using the methodology disclosed in U.S. Pat.No. 5,023,264, which is hereby incorporated by reference; and asdescribed in Example 21.

The linker group attached to rapamycin can be coupled to a second linkergroup using standard methodology described in the peptide literature;typically by activating the electrophilic moiety, with DCC type couplingreagent, or with N-hydroxysuccinimide, or as an activated ester oranhydride. The activated electrophilic end of one linking moiety canthen be reacted with the nucleophilic end of the other linker moiety.

The coupling of the rapamycin linking group moiety to the immunogeniccarrier can be accomplished under standard literature conditions. Ingeneral, for reaction with a nucleophilic group on the immunogeniccarrier material, an electrophilic moiety, such as a carboxylic acid, onthe linking group is activated with a suitable activating agent such asN-hydroxysuccinimide, and then reacted with the nucleophilic moiety onthe immunogenic carrier material. Examples 2 and 3 specificallyexemplify this technique. Similar methodology is employed for thecoupling of a nucleophilic moiety on the linking group to anelectrophilic moiety on the immunogenic carrier material. In such cases,the electrophilic moiety on the immunogenic carrier material isactivated as described above, and then reacted with the nucleophilic endof the linking group.

The reagents used to prepare the compounds of the invention arecommercially available or can be prepared by methods that are disclosedin the literature.

This invention also covers analogous conjugates of other rapamycins suchas, but not limited to, 29-demethoxyrapamycin, [U.S. Pat. No. 4,375,464,32-demethoxyrapamycin under C.A. nomenclature]; rapamycin derivatives inwhich the double bonds in the 1-, 3-, and/or 5-positions have beenreduced [U.S. Pat. No. 5,023,262]; 42-oxorapamycin [U.S. Pat. No.5,023,262]; 27-oximes of rapamycin [U.S. Pat. No. 5,023,264];27-hydrazones of rapamycin [U.S. Pat. No. 5,120,726];29-desmethylrapamycin [U.S. Pat. No. 5,093,339, 32-desmethylrapamycinunder C.A. nomenclature]; 7,29-bisdesmethylrapamycin [U.S. Pat. No.5,093,338, 7,32-desmethylrapamycin under C.A. nomenclature]; and15-hydroxy- and 15,27-bishydroxy-rapamycin [U.S. Pat. No. 5,102,876].The disclosures in the above cited U.S. patents are hereby incorporatedby reference. Also covered are conjugates of the rapamycin 1,3-DielsAlder adduct with diethyl azidodicarboxylate and rapamycin 1,3-DielsAlder adduct with phenyltriazoline dione. The preparation of thesecompounds is described in Examples 14 and 15.

The compounds of this invention are rapamycin immunogen, detector, andmatrix bound conjugates that are useful for the generation and detectionof antibodies specific for rapamycin and derivatives thereof, formeasuring levels of rapamycin or a derivative thereof in biological orlaboratory fluids, and for isolating rapamycin binding proteins.Rapamycin derivatives as defined here are compounds containing arapamycin nucleus in which one or more of the hydroxyl groups has beenesterified into a carboxylic ester, a carbamate, a sulfonate ester, anamide, or the like, or one or more of the ketones has been reduced to ahydroxyl group, or one or more of the double bonds has been reduced, orone ketones has been converted to an oxime or a hydrazone. Otherrapamycin derivatives for which the compounds of this invention can beused for measuring levels of or generating antibodies to will beapparent to one skilled in the art based on this disclosure.

Antibodies specific for rapamycin using the rapamycin immunogenconjugates of this invention may be generated by standard techniquesthat are known in the art. Typically, a host animal is inoculated at oneor more sites with the immunogen conjugate, either alone or incombination with an adjuvant. The typical host mammals include, but arenot limited to, mice, goats, rabbits, guinea pigs, sheep, or horses.Subsequent injections can be made until a sufficient titer of antibodiesare produced. The antibodies generated from the rapamycin immunogenconjugates of this invention can be used in numerous immunoassays, fordetermining rapamycin levels, in ELISAs, radioimmunoassays, inchemiluminesence immunoassays, and in fluorescent immunoassays. Althoughmany variations of the immunoassay can be used (antigen capture,antibody capture, competitive inhibition, or two antibody immunoassay),a basic competitive inhibition immunoassay can be performed as follows:Antibody specific for the ligand is usually bound to a matrix. Asolution is applied to decrease nonspecific binding of the ligand to thematrix. After rinsing the excess away, the antibody coupled matrix maybe treated in some cases so it can be stored. In a competitiveinhibition assay, the ligand standard curve is made and added with therapamycin detector conjugate to compete for binding to therapamycin-specific antibody. If necessary, the excess is removed. Thedetector molecule is detected by the standard methods used by oneskilled in the art. Different formats can be used, which include but arenot limited to, dipstick assays, FPIA, EMIT, ELISA, VISTA, RIA, andMEIA. Detector conjugates of the present invention can be prepared touse in the above assays. For example, the detector conjugates can beCarrier material with labeled fluorescent, chemiluminescent, orenzymatic moieties.

This invention also provides for the use of the rapamycin immunogenconjugates or rapamycin specific antibodies in a test kit that can becommercially marketed. The test kit may be used for measuring levels ofrapamycin in biological or laboratory fluids. Test kit components mayinclude rapamycin antibodies, antisera, or rapamycin carrier conjugates.The conjugates or antibodies may be bound to a solid matrix, andrapamycin derivatives or antibodies may be radiolabeled if the assay sorequires. Standard concentrations of rapamycin can be included so that astandard concentration curve can be generated. Suitable containers,microtiter plates, solid supports, test tubes, trays, can also beincluded in any such kit. Many variations of reagents can be included inthe kit depending on the type of assay used.

The following is illustrative of the use of a rapamycin immunogenconjugate of this invention to generate rapamycin specific antibodiesand detect them using an ELISA format immunoassay. Five mice wereimmunized with 50 μg rapamycin 31,42-diester with glutaric acidconjugate with keyhole limpet hemocyanin in Complete Freund's Adjuvantintrasplenically and after about one month were boosted with 50 μg ofrapamycin 31,42-diester with glutaric acid conjugate with keyhole limpethemocyanin in incomplete Freund's Adjuvant into the footpads. Microtiterplates (IMMUNOLON I) were coated overnight with 100 μl of goatanti-mouse antibody (10 μg/ml in 10 mM potassium phosphate buffer, pH7.2) at 4° C. The plates were flicked and blocked with 100 μl of 1%bovine sera albumin in phosphate buffered saline overnight at 4° C.After flicking and washing the plates thrice with 10 mM phosphatebuffer, pH 7.05, 30 mM NaCl, 0.02% TRITON X-100 (polyethylene glycoltert-octylphenyl ether), and 0.004% thimerosal wash buffer, 100 μl ofeach mouse sera diluted with phosphate buffer solution was added to awell and incubated at room temperature for overnight. After flicking andwashing the plates thrice with wash buffer, rapamycin 31,42-diester withglutaric acid conjugate with horseradish peroxidase (compound of Example10 (100 μl, 0.5 ng/ml) was added and incubated for 1 hour at roomtemperature in the dark. After flicking and washing the plates thricewith wash buffer, tetramethyl benzidine (TMB) substrate with H202 wasadded and the plates were incubated covered for 30 mm. at roomtemperature in the dark. The optical density was read on aspectrophotometer at 450 nm. As shown in Table I, five of the five micehad antibodies reactive for rapamycin 31,42-diester with glutaric acidconjugate with horseradish peroxidase (compound of Example 10).

TABLE I MOUSE # DILUTION^(a) O.D. 6902 1/300 0.199 6903 1/100 0.231 69041/500 0.412 6905 1/100 0.121 6906 1/300 0.321 background — 0.076^(a)Dilution of mouse sera in PBS

The results in Table 1 show that mouse 6904 produced the most antibodiesto the compound of Example 10. Hybridomas were generated using standardmethodology. Following a splenectomy of a mouse immunized and boosted 3times with the compound of Example 4, spleen cells were fused to SP20cells to produce hybridomas. The hybridomas were evaluated for theproduction of rapamycin specific antibodies using an ELISA assay asbriefly described below.

Microtiter plates (IMMUNOLON I) were coated overnight with 100 μl ofgoat anti-mouse antibody (10 μg/ml in 10 mM potassium phosphate buffer,pH 7.2) at 4° C. The plates were flicked and blocked with 100 μl of 1%bovine sera albumin in phosphate buffered saline (PBS) overnight at 4°C. After flicking and washing the plates thrice with 0.2×PBS containing0.02% TRITON X-100 and 0.004% thimerosal, 100 μl of each hybridomasupernatant was added to a well and incubated at room temperature forovernight. After flicking and washing the plates thrice with 0.2×PBScontaining 0.02% TRITON X-100 and 0.004% thimerosal, the compound ofExample 22 (100 μl, 0.17 μM) was added and incubated for 1 hour at 4° C.After flicking and washing the plates thrice with 0.2×PBS containing0.02% TRITON X-100 and 0.004% thimerosal, strepavidin or avidinconjugated to horseradish peroxidase (100 μl, 0.2 μg/ml) was added andincubated at room temperature for 1 hour in the dark. After flicking andwashing the plates thrice with 0.2×PBS containing 0.02% TRITON X-100 and0.004% thimerosal, TMB substrate and H₂O₂ was added and the plates wereincubated covered for 30 mm. at room temperature in the dark. Theoptical density was read on a spectrophotometer at 450 nm. An opticaldensity reading of 0.25-3 indicates specific antibody binding. Theresults in Table 2 show that the hybridoma from well P4G1 is positivefor binding to the compound of Example 22, and is therefore specific forrapamycin.

TABLE 2 Screening for Rapamycin Specific Monoclonal Antibodies WELLOPTICAL DENSITY P3H4 0.120 P3H5 0.105 P4G1 1.940

The hybridoma cell line in P4G1 was cloned by limiting dilution and isdesignated as hybridoma cell line, RAP-42-OVAF₂#1hc. Therapamycin-specific antibody, designated as RAP-42-OVAF₂#1MoAb, wasisolated and purified using conventional methodology. HybridomaRAP-42-OVAF₂#1hc was deposited under the terms of the Budapest Treaty atthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, USA, on Mar. 10, 1994, and was grantedaccession number NB 11568.

The compounds of Examples 12 and 13 can be used in an assay for thedetection of polyclonal antibodies and monoclonal antibodies specificfor rapamycin as described below.

Microtiter plates (IMMUNOLON I) were coated overnight with 100 μl ofgoat anti-mouse antibody (10 μg/ml in 10 mM potassium phosphate buffer,pH 7.2) at 4° C. The plates were flicked and blocked with 100 μl of 1%bovine sera albumin in phosphate buffered saline overnight at 4° C.After flicking and washing the plates thrice with wash buffer, 100 μl ofrabbit sera diluted 1:5 in phosphate buffered saline was added to a welland incubated at room temperature for overnight. After flicking andwashing the plates thrice with wash buffer, rapamycin 42-ester with3-[3-(4-imino-butylthio)succinimidyl]phenacylglycine conjugate withhorseradish peroxidase (compound of Example 12) (100 μl, 0.5 ng/ml) orrapamycin 42 ester with (N-(3-carboxyphenyl)-3-thiosuccinimidyl)glycineconjugate with horseradish peroxidase (compound of Example 13) (100 μl,0.5 ng/ml) was added and incubated for 1 hour at room temperature in thedark. After flicking and washing the plates thrice with wash buffer, TMBsubstrate with H₂O₂ was added and the plates were incubated covered for30 mm. at room temperature in the dark. The optical density was read ona spectrophotometer at 450 nm. The results are shown in Table III.

TABLE 3 Comparison of Anti-rapamycin Antibody Levels in RabbitsImmunized with the Compound of Example 3 vs. Naive Rabbits Using aCapture ELISA Assay Prebleed ΔA450 (3rd Bleed-Prebleed) Rabbit No.Example 10 Example 10 Example 12 Example 13 81 0.119 0.713 0.217 0.11489 0.136 0.037 0.026 0.020

The data in Table 3 show that the compounds of Examples 12 and 13 can beused to detect antibodies specific for rapamycin in an a mammal, as seenin rabbit number 81.

The following is an example of the measurement of rapamycinconcentrations using a competitive inhibition assay for rapamycin withan ELISA format using an antibody specific for rapamycin. Microtiterplates (IMMUNOLON I) were coated overnight with 100 μl of goatanti-mouse antibody (10 μg/ml in 10 mM potassium phosphate buffer, pH7.2) at 4° C. The plates were flicked and blocked with 100 μl of 1%bovine sera albumin in phosphate buffered saline overnight at 4° C.After flicking and washing the plates thrice with wash buffer, therapamycin specific antibody described above (100 μl of 1 μg/ml) wasadded to each well and incubated at room temperature for 1-4 hour. Afterflicking and washing the plates thrice with wash buffer, rapamycin31,42-bis(hemiglutarate) conjugate with horseradish peroxidase (100 μl,0.5 ng/ml) was added and incubated for 1 hour at room temperature in thedark. After flicking and washing the plates thrice with wash buffer, TMBsubstrate was added and the plates were incubated covered for 5 mm atroom temperature in the dark. The optical density was read on aspectrophotometer at 450 nm. Results of the competition betweenrapamycin and rapamycin 31,42-diester with glutaric acid conjugate withhorseradish peroxidase binding to mouse sera are shown in Table 4. Fromthese results, a standard curve can be constructed and the concentrationof rapamycin in a sample can be determined.

TABLE 4 Free OPTICAL DENSITY x1000 RAPAMYCIN 1 2 Avg % Inhibition 10 μM158 158 158 74.1 5 182 194 188 69.2 0.5 304 322 313 48.6 0.05 494 501498 18.4 0.005 528 546 537 11.9 0.0005 601 611 606 0.6 0 583 636 610 —

The compound of Example 11 (rapamycin 42-ester withN-[9H-fluoren-9-ylmethoxy)carbonyl]glycine) can be deprotected by theprocedure used in Example 12 (to give rapamycin 42-ester with glycine)and conjugated to a solid matrix. It can bind rapamycin specificantibodies as used in some dipstick immunoassay methods or to isolaterapamycin binding proteins. The following example illustrates that 803resonance units (RU) of the compound of Example 11 can be immobilized ona solid matrix using the BIAcore's standard protocol based on EDC andNHS used in a BIAcore. This matrix bound 1401 RU units of rapamycinspecific antibody. The kinetics of association and dissociation weredetermined for each concentration of antibody tested (0.625, 1.25, 2.5,5.0, 10.0 ug/ml). These data show that the compound of Example 11, evenwhen bound to a matrix was accessible to binding by a rapamycin-specificantibody and the interaction could be characterized. Similar procedurescan be used to bind a rapamycin-binding protein to deprotected rapamycin42-ester with N-[9H-fluoren-9-ylmethoxy)carbonyl]glycine conjugatedmatrix. This matrix can also be used for the isolation of novel bindingproteins, as practiced by one skilled in the art. Deprotected rapamycin42-ester with N-[9H-fluoren-9-ylmethoxy)carbonyl]glycine can be used toisolate binding proteins of rapamycin-FKBP complex by one of thefollowing methods. In one approach, tissue or cell lysates containingthe appropriate protease inhibitors are incubated with FKBP which hasbeen incubated with a deprotected-rapamycin 42-ester withN-[9H-fluoren-9-ylmethoxy)carbonyl]glycine conjugated matrix for asufficient time to allow binding. Various buffers are used to rinse theproteins which are nonspecifically bound. Proteins are released by theaddition of additional buffers which disrupt the bond between therapamycin nucleus-FKBP and the binding proteins.

The following examples represent the preparation of representativecompounds of this invention.

EXAMPLE 1 Rapamycin 42-ester with Succinic Acid

1.1 g (11 mmol) of succinic anhydride and 400 mg ofdimethylaminopyridine (DMAP) were added to a stirring solution of 5 g(5.5 mmol) of rapamycin and 880 μl of pyridine in 15 ml of methylenechloride. The reaction mixture was stirred for 2 days at roomtemperature, diluted with methylene chloride and washed with three 50 mlportions of 1N HCl. The organic layer was then dried over Na₂SO₄ andconcentrated in vacuo affording crude product. Pure material wasobtained by reverse phase HPLC with 55% acetonitrile/water as eluantaffording 1 g (18%) of the title compound. Spectral data follows: ¹H NMR(CDCl₃, 300 MHz) 4.650 (m, 1H, H₂COC═O), 4.168 (d, 1H, H₂COH), 2.795 (s,4H, OC═OCH₂CH₂C═O).

EXAMPLE 2 Rapamycin 42-ester with (N-hydroxysuccinimide(hemisuccinate))

21 mg (0.098 mmol) of DCC and 12 mg (0.098 mmol) of N-hydroxysuccinimidewere added to a stirring solution of 100 mg of rapamycin 42-ester withsuccinic acid in 3 ml ethyl acetate. The reaction mixture was stirredovernight at room temperature, filtered, and concentrated in vacuoaffording crude product. Pure material was obtained by reverse phaseHPLC with 80% acetonitrile/water as eluant affording 75 mg (69%) of thetitle compound. Spectral data follows: ¹H NMR (CDCl₃, 300 MHz) 4.650 (m,1H, H₂COC═O), 4.168 (d, 1H, H₂COH), 2.951 (m, 2H, OC═OCH₂), 2.795 (m,4H, OC═OCH₂CH₂C═O), 2.705 (m, 2H, OC═OCH₂); MS (neg.ion FAB) 1110 (M⁻),1056, 1012, 913, 148 (100).

EXAMPLE 3 Rapamycin 42-ester with Succinic Acid Conjugate with KeyholeLimpet Hemocyanin

197 mg of keyhole limpet hemocyanin in 6 ml of 0.05 M phosphate bufferwas added to a stirring solution of 37 mg of rapamycin 42-ester with(N-hydroxysuccinimide(hemisuccinate)) in 3 ml of 1,4 dioxane and thereaction was left stirring for 3 days at 4° C. The reaction mixture wasthen dialyzed for 24 hr at 4° C. in 1500 ml of 0.05 M phosphate bufferto give the title compound which could be used without furtherpurification. The number of rapamycin 42-ester with succinic acidmoieties per keyhole limpet hemocyanin was approximately 42:1.

EXAMPLE 4 Rapamycin 42-ester with Succinic Acid Conjugate with Ovalbumin

197 mg of ovalbumin in 6 ml of 0.05 M phosphate buffer was added to astirring solution of 37 mg of rapamycin 42-ester with(N-hydroxysuccinimide(hemisuccinate)) in 3 ml of 1,4 dioxane and thereaction was left stirring for 3 days at 4° C. The reaction mixture wasthen dialyzed for 24 hr at 4° C. in 1500 ml of 0.05 M phosphate bufferto give the title compound which could be used without furtherpurification.

EXAMPLE 5 Rapamycin 42-ester with Succinic Acid Conjugate withHorseradish Peroxidase

16 mg of horseradish peroxidase in a solution of 0.4 ml of 1,4 dioxaneand 0.4 ml of 0.5% sodium bicarbonate was added to 1 mg of rapamycin42-ester with (N-hydroxysuccinimide(hemisuccinate)) in 40 μl of 1,4dioxane and the reaction left stir for 2.5 hr at 4° C. The reactionmixture was then dialyzed for 24 hr at 4° C. in 1500 ml of 0.05 Mphosphate buffer to give the title compound which could be used withoutfurther purification.

EXAMPLE 6 Rapamycin 31,42 Diester with Glutaric Acid

The title compound was prepared according to the method used in Example1.

EXAMPLE 7 Rapamycin 31,42-diester with(N-hydroxysuccinimide(hemiglutarate))

To a solution of 15.9 mg of rapamycin 31,42-diester with glutaric acidin 160 μL of dimethyl formamide was added 3.65 mg ofN,N-dimethylaminopropyl-ethylcarbodiimide and 1.8 mg ofN-hydroxysuccinimide. The reaction mixture was allowed to stir untilreaction was complete, poured into water, and extracted with ethylacetate. The organic layers were combined, dried over sodium sulfate,filtered, and concentrated in vacuo to give the title compound, whichwas stored at 4° C. at 0.1 N sodium phosphate buffer and used withoutfurther purification.

EXAMPLE 8 Rapamycin 31,42-diester with Glutaric Acid Conjugate withKeyhole Limpet Hemocyanin

To 20 mg of keyhole limpet hemocyanin in 2 mL of 0.1 M NaHCO₃ was added55 μL of rapamycin 31,42-diester with(N-hydroxysuccinimide(hemiglutarate)) at 0° C. in 10 μL increments overa 30 min period. The solution was gently shaken until reaction wascomplete, centrifuged at 6000 rpm for 20 min, and unconjugated startingmaterial was separated from the title compound on a G-25 column withphosphate buffer solution. The conjugate was mixed with glycerol at 50%and stored at −70° C. The number of rapamycin 31,42-diester withglutaric acid moieties per keyhole limpet hemocyanin ranged from17-45:1.

EXAMPLE 9 Rapamycin 31,42-diester with Glutaric Acid Conjugate withOvalbumin

To 20 mg of ovalbumin in 2 mL of 0.1 M NaHCO₃ was added 55 μL ofrapamycin 31,42-diester with (N-hydroxysuccinimide(hemiglutarate)) at 0°C. in 10 μL increments over a 30 min period. The solution was gentlyshaken until reaction was complete, centrifuged at 6000 rpm for 20 min,and unconjugated starting material was separated from the title compoundon a G-25 column with phosphate buffer solution. The conjugate was mixedwith glycerol at 50% and stored at −70° C.

EXAMPLE 10 Rapamycin 31,42-diester with Glutaric Acid Conjugate withHorseradish Peroxidase

To 10 mg of horseradish peroxidase in 1 mL of 0.1 M NaHCO₃ was added 105μL of rapamycin 31,42-diester with (N-hydroxysuccinimide(hemiglutarate))in 10 μL increments over a 30 min period. The solution was gently shakenuntil complete, centrifuged at 6000 rpm for 20 min, and eluted from aG-25 column with phosphate buffer solution. The conjugate was mixed withglycerol at 50% and stored at −20° C.

EXAMPLE 11 Rapamycin 42-ester withN-[9H-fluoren-9-ylmethoxy)carbonyl]glycine

To a chilled (0° C.) solution of rapamycin (0.73 g, 0.08 mmol) inmethylene chloride (5 mL) was added 0.6 g (1.19 mmol) ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]glycine pentafluorophenyl ester,followed by pyridine (0.85 mL, 10.5 mmol) and dimethylaminopyridine (18mg, 0.14 mmol) to form a heterogeneous solution, which becamehomogeneous upon warming to room temperature. The reaction mixture wasstirred at room temperature overnight. A large excess of EtOAc wasadded. The organic layer was washed with 0.5 N HCl (2×) and brine, dried(MgSO₄), and concentrated to yield an off-white foam. Flashchromatography (30-50% hexane/EtOAc) yielded the title compound in 71%yield (0.679 g, 0.57 mmol). Mass spec (negative ion FAB) M⁻ at m/z 1192.

EXAMPLE 12 Rapamycin 42-ester with3-[3-(4-iminobutylthio)succinimidyl]phenacylglycine Conjugate withHorseradish Peroxidase

To a solution of rapamycin 42-ester withN-[9H-fluoren-9-ylmethoxy)carbonyl]glycine (10 mg, 8.4 μmol) inacetonitrile (84 μL) was added 10 μL (in acetonitrile at 0.84 M) ofdiethylamine. The reaction mixture was stirred at room temperature for60 minutes and the solvent was removed with a stream of nitrogen. Theresidue was dissolved in acetonitrile (100 μL) and washed with hexane (5times, 200 μL), followed by concentration of the solvent with a nitrogenstream. The resulting rapamycin 42-ester with glycine was taken up in asolution of m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) (2 mg) in DMF(200 μL) and allowed to incubate for two hours at 4° C., followed by theaddition of 50 nM ethanolamine (20 μL) in 50 mM Tris HCl, pH 8.0.Horseradish peroxidase (5 mg) and Rabbit IgG (10 mg) were treated with2-iminothiolane and purified with Sephadex G-25, followed by theaddition of the MBS-rapamycin glycine ester adduct. The mixture wasincubated overnight at 4° C. and purified by gel filtration on SephadexG-25 to provide the title compound.

EXAMPLE 13 Rapamycin 42 Ester with(N-(3-carboxyphenyl)-3-thiosuccinimidyl)-glycine Conjugate withHorseradish Peroxidase

To a solution of rapamycin 42-ester withN-[9H-fluoren-9-ylmethoxy)carbonyl]glycine (10 mg, 8.4 μmol) inacetonitrile (84 μL) was added 10 μL (in acetonitrile at 0.84 M) ofdiethylamine. The reaction mixture was stirred at room temperature for60 minutes and the solvent was removed with a stream of nitrogen. Theresidue was dissolved in acetonitrile (100 μL) and washed with hexane (5times, 200 μL), followed by concentration of the solvent with a nitrogenstream. The resulting rapamycin 42-ester with glycine was taken up in asolution of N-succinimidyl S-acetylthioacetate (2 mg) in DMF (200 μL).The reaction mixture was stirred at room temperature for 15 minutes andthen at 4° C. overnight. A solution of hydroxylamine HCl (7 mg in 50 μLDMF) was added to the solution of rapamycin reaction mixture, incubatedfor one hour, followed by the addition of MBS-horseradish peroxidaseadduct and MBS-Rabbit IgG to give the title compound which was purifiedby Sephadex G-25 gel filtration.

EXAMPLE 14 Rapamycin 1,3, Diels Alder Adduct with DiethylAzidodicarboxylate

Rapamycin (1 g, 1.093 mmol) and diethyl azodicarboxylate (0.381 g, 2.187mmol) were dissolved in dichloromethane (10 ml) and heated at 65° C.overnight, TLC showed that the reaction was complete. The mixture waspurified on a silica gel column using ethyl acetate as eluant to providea white solid (0.750 g) which was triturated with hexane and air driedto give the title compound (0.666 g) as a powder.

Anal Calc for C₅₇H₈₉N₃O₁₇: C, 62.91; H, 8.24; N, 3.86. Found: C, 62.81;H, 8.12; N, 3.91. IR (KBr, cm⁻¹) 3450, 1720 NMR (CDCl₃) δ 6.15 (m, 1H),5.20 (d, 1H), 3.40 (s, 3H), 3.30 (s, 3H), 3.15 (s, 3H), 0.9 (t, 3H),0.72 (q, 1H) MS (−FAB) 1087 (M⁻)

EXAMPLE 15 Rapamycin 1,3, Diels Alder Adduct with Phenyltriazolinedione

Rapamycin (0.66 g, 721 mmol) was dissolved in dichloromethane (10 ml)and cooled to 0° C. To this was added, dropwise, a solution ofphenyltriazolinedione (0.133 g, 758 mmol) in dichloromethane (10 ml).The solution was stirred overnight, TLC showed the reaction was notcomplete. Additional phenyltriazenedione (0.025 g, 27 mmol) was added.The reaction was purified using HPLC (4.1×31 cm, SiO₂) with ethylacetate as eluant to provide the title compound as a solid. The solidwas triturated with 30 ml of hexane and 1 ml of ethyl acetate filteredand air dried to give the title compound as a powder (0.383 g).

Anal Calc for C₅₉H₈₄N₄O₁₅: C, 65.05; H, 7.77; N, 5.14. Found: C, 65.39;H, 7.98; N, 4.92. IR (KBr, cm⁻¹) 3450, 1715 NMR (DMSO) δ 7.50 (m, 3H),7.40 (m, 2H), 3.11 (s, 3H), 3.00 (s, 3H) 2.95 (s, 3H), 0.8 (q, 1H) MS(−FAB) 1088 (M⁻)

The following are representative examples of fluorescent rapamycinderivatives that can be conjugated via a linker at the 31-position ofrapamycin.

EXAMPLE 16 42-Dansylrapamycin

Rapamycin (200 mg, 0.22 mmol) in dry pyridine (2 ml) was cooled to 0° C.and was treated with dansyl chloride (840 mg, 3.1 mmol). The reactionwas warmed to room temperature and stirred for 24 hours. The reactionmixture was poured into cold 2N HCl (30 ml) and was extracted with ethylacetate (4×25 ml). The ethyl acetate was pooled and washed with brine,dried over MgSO₄, filtered and concentrated in vacuo. The residue waschromatographed on silica with 25% ethyl acetate in benzene. Thisafforded 150 mg of the title compound as a yellow powder, mp 101-104° C.

EXAMPLE 17 Rapamycin 42-ester with Pyrene Butyric Acid

Rapamycin (459 mg, 0.5 mmol) and pyrenebutyric acid (216 mg, 0.75 mmol)were dissolved in THF/CH₂Cl₂ (10 ml, 1:1).1-(3-Dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (146 mg,0.67 mmol) and 4-dimethylaminopyridine (15 mg) were added to thesolution. The reaction was allowed to warm to room temperature over 15hours. The reaction was diluted with CH₂Cl₂ and washed with 5% HCl, thenbrine. The solution was dried over MgSO₄, filtered and evaporated to asolid. The solid was applied to a 3 mm silica gel Chromatron plate whichwas eluted with 50% ethyl acetate in hexane to provide 180 mg of thetitle compound as a foam. The reaction also afforded 100 mg of31,42-diesterified rapamycin.

IR (KBr, cm⁻¹) 3420, 1740 NMR (CDCl₃) d 8.3 (d, 1H), 8.14 (dd, 2H), 8.10(d, 2H), 7.85 (d, 1H), 3.34 (s, 3H), 3.30 (s, 3H). 3.11 (s, 3H) MS(−FAB) 1183 (M⁻)

The following are representative examples of rapamycin derivatives thatcan be conjugated to immunogenic carriers by the procedures describedabove or can be connected to another linker and then conjugated.

EXAMPLE 18 Rapamycin 42-carbomethoxymethyl Ether and Rapamycin42-bis(carbomethoxymethyl ether)

Rapamycin (2.0 g, 2.187 mmol) and rhodium (II) acetate (0.37 g, 0.08mmol) were heated to reflux in benzene and treated with a solution ethyldiazoacetate (500 ml) in benzene (10 ml) over 10 minutes. The solutionwas cooled to room temperature and was stirred overnight. TLC showedthat the reaction was incomplete. Two additional portions ofethyldiazoacetate (3 ml) were added at 24 hour intervals. The mixturewas concentrated and purified by flash chromatography over silica usingethyl acetate. This provided the 42-monoether (1 g) and the 31,42diether (0.850 g) as oils. The 42-monoether was triturated in a mixtureof hexane, ethyl acetate and dichloromethane over the weekend to givethe product as a powder. The diether was purified on HPLC on a silicagel column with ethyl acetate as eluant. This provided the product as asolid.

Analytical data for the monoether:

Analysis Calc for C₅₅H₈₅NO₁₅: C, 66.04; H, 8.57; N, 1.40. Found: C,65.29; H, 8.64; N, 1.60. IR (KBr, cm⁻¹) 3420, 1715 NMR (CDCl₃) d 4.82(s, 1H), 3.41 (s, 3H), 3.33 (s, 3H), 3.13 (s, 3H), 1.28 (t, 3H), 0.70(q, 1H) MS (−FAB) 999 (M⁻)

Analytical data for the diether:

Analysis Calc for C₅₉H₉₁NO₁₇: C, 65.23; H, 8.44; N, 1.29. Found: C,63.29; H, 8.40; N, 1.44. IR (KBr, cm⁻¹) 1740 NMR (CDCl₃) δ 6.36 (q, 2H),5.24 (s, 1H), 3.39 (s,3H), 3.32 (s,3H), 3.12 (s, 3H), 0.65 (q, 1H) MS(−FAB) 1085 (M⁻)

EXAMPLE 19 Rapamycin 42-(4-nitrophenyl)carbonate and Rapamycin31,42-bis(4-nitrophenyl)carbonate

Rapamycin (0.450 g, 0.49 mmol) was dissolved in dry dichloromethane (10ml) and cooled to 0° C. To this solution was added pyridine (0.4 ml, 5.7mmol) and a crystal of 4-dimethyl aminopyridine. A solution of4-nitrophenyl chloroformate (0.3 g 1.49 mmol) in dichloromethane (3 ml)was added. The solution was allowed to warm to room temperatureovernight and was stirred at room temperature for 24 hours. The reactionwas quenched into 0.1N HCl (5 ml) and the aqueous layer was washed withdichloromethane. The organic layer was dried over MgSO₄, filtered, andevaporated in vacuo to afford a yellow solid. Chromatography over silicagel with 75% Ethyl acetate in hexane afforded 180 mg of the42-monocarbonate and 47 mg of the 31,42-dicarbonate as yellow solids.

EXAMPLE 20 42-O-(Phenoxythiocarbonyl)-rapamycin

Rapamycin (1.030 g, 1.12 mmol) was dissolved in dry dichloromethane (100ml) and was cooled to 0° C. To this solution was added pyridine (0.27ml, 3.33 mmol) and a crystal of 4-dimethyl aminopyridine. A solution ofthiophenyl chloroformate (0.47 ml 1.49 mmol) in dichloromethane (5 ml)was added to the reaction mixture. The solution was allowed to warm toroom temperature overnight and was stirred at room temperature for 24hours. The reaction was quenched into 0.1N HCl (5 ml) and the aqueouslayer was washed with dichloromethane. The organic layer was dried overMgSO₄, filtered and evaporated in vacuo to afford a yellow solid.Chromatography on a 4 mm silica gel Chromatotron plate with a gradientof 40% to 70% ethyl acetate in hexane afforded 520 mg of the titlecompound as a yellow foam.

Analysis Calc for C₅₈H₈₃NOS₁₄: C, 66.32; H, 7.97; N, 1.33. Found: C,66.48; H, 8.05; N, 1.12. IR (KBr, cm⁻¹) 3420, 1715 NMR (CDCl₃) δ 7.41(t, 1H), 7.25 (t, 2H), 7.12 (d, 1H), 3.45 (s, 3H), 3.33 (s, 3H), 3.13(s, 3H) MS (−FAB) 1049 (M⁻)

EXAMPLE 21 Rapamycin-O-carboxymethyl-27-oxime

To a solution of 600 mg (650 μM) of rapamycin in 6 mL of methanol wasadded at room temperature, 100 mg (1.2 mmol) of anhydrous sodium acetateand 140 mg (660 μM) of carboxymethoxylamine hemihydrochloride. Afterstirring overnight at room temperature, the reaction was complete. Thereaction mixture was concentrated in vacuo and the residue wastriturated with water. The solids were filtered and washed thoroughlywith water. The product was dried under high vacuum to give 575 mg(89.7%) of a white solid. ¹³C and ¹H NMR indicated a mixture of E and Zisomers for the oxime derivative at position 27.

¹H NMR (CDCl₃, 400 MHz): 3.43 and 3.41 (2s, 3H, CH₃O), 3.30 (s, 3H,CH₃O), 3.18 and 3.12 (2s, 3H, CH₃O), 1.82 (s, 3H, CH₃C═C), 1.695 and1.633 (2s, 3H, CH₃C═C); ¹³C NMR (CDCl₃, MHz): 215.8 (C═O), 211.5 (C═O),194.5 (C═O), 191.0 (C═O), 172.5 (C═O), 169.0 (C═O), 168.5 (C═O), 167.0(C═O), 161.5 (C═NOC), 160.0 (C═NOC), 140.0; MS (neg. ion FAB: 985(M−H)⁻, 590, 167, 128, 97, 75 (100%)

Analysis Calcd for C₅₃H₈₂N₂O₁₅.0.15 H₂O: C, 63.90; H, 8.40; N, 2.81.Found: C, 63.81; H, 8.41; N, 2.85.

The following compound was used in the generation of rapamycin specificantibodies.

EXAMPLE 22 Rapamycin 42-ester with Glycylbiotin

To a solution of biotin (0.83 g, 3.4 mmol) in 60 mL of DMF was addedglycine t-butyl ester hydrochloride (0.57 g, 3.4 mmol),N-methylmorpholine (0.92 mL, 8.36 mmol), 1-hydroxybenzotriazole (0.61 g,3.99 mmol) and 1-(3-Dimethylaminopropyl)-3-ethylcarbo-diimidehydrochloride (0.65 g, 3.4 mmol). The reaction mixture was stirred atroom temperature for 7 days. The DMF was concentrated, ethyl acetate wasadded, and the organic layer was washed with water, 0.5 N HCl, saturatedsodium. bicarbonate, and brine. The ethyl acetate layer was dried(MgSO₄) and concentrated to yield tert-butylglycylbiotin as a whitesolid which was primarily one spot on TLC (0.611 g, 1.71 mmol, 50%).Mass spec [M+H]⁺ at m/z 358.

To a solution of tert-butylglycylbiotin (0.271 g, 0.758 mmol) in CH₂Cl₂(0.5 mL) was added 0.5 mL trifluoroacetic acid. The reaction mixture wasstirred at room temperature for 2 h, concentrated, and triturated withanhydrous diethyl ether. The off-white precipitate was collected toyield 0.209 g (0.694 mmol, 92%) of glycylbiotin. Mass spec [M+H]⁺ at m/z302.

To a solution of glycylbiotin (0.65 g, 2.16 mmol) in1-methylpyrrolidinone (5 mL) was added 6 mL of CH₂Cl₂, causing aprecipitate to form which persisted even after the addition of 0.33 mL(2.36 mmol) of triethylamine. To this heterogenous solution was added 2g (2.19 mmol) of rapamycin, 0.43 g (2.24 mmol) of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 30 mg(2.46 mmol) of DMAP. After several hours, the reaction mixture becamehomogenous, and was stirred an additional four days. A large excess ofethyl acetate was added and the organic layer was washed with water, 0.5N HCl, saturated sodium bicarbonate, and brine. The organic layer wasdried (MgSO₄) and concentrated. The light yellow foam was trituratedwith hot anhydrous diethyl ether to yield 1.2 g of impure title compoundas a light yellow solid. A portion (0.5 g) of this material was flashchromatographed in 5% MeOH/CHCl₃, and triturated again in hot ether toyield 87 mg of the title compound contaminated with a small amount ofrapamycin. This material was rechromatographed (gradient 0-5%MeOH/CHCl₃), and triturated a final time with ether to yield 34 mg(0.028 mmol) of pure title compound as a white solid. Mass spec,negative FAB M⁻ at m/z 1196.

1. A rapamycin conjugate represented by formula I, or a salt thereof:

wherein R¹ and R² are each, independently, hydrogen or —(R³-L-R⁴)_(a)—; L is a linking group; R³ is selected from the group consisting of carbonyl, —S(O)—, —S(O)₂, —P(O)₂—, —P(O)(CH₃)—, —C(S)—, and —CH₂C(O)—; R⁴ is a selected from the group consisting of carbonyl, —NH—, —S—, —CH₂—, and —O—; a=1-5; x=0-1; y=0-1; z is from about 1 to about 120; and Carrier is immunogenic carrier material, detector carrier material, or a solid matrix, with the proviso that R¹ and R² are both not hydrogen; and further provided that when a is greater than 1, each L group can be the same or different; and still further provided that x is 0 if R¹ is hydrogen and y is 0 if R² is hydrogen, and if x and y are both 1, the Carrier moiety is the same in both cases.
 2. A rapamycin conjugate represented by formula II, or a salt thereof:

wherein R¹ and R² are each, independently, hydrogen or —R³-L-R⁴—; L is -A-(CR⁵R⁶)_(b)[B—(CR⁷R⁸)_(d)]_(e)—; A is —CH₂— or —NR⁹—; B is —O—, —NR⁹—, —S—, —S(O)—, or —S(O)₂—; R³ is selected from the group consisting of carbonyl, —S(O)—, —S(O)₂—, —P(O)₂—, —P(O)₂(CH₃)—, —C(S)—, and —CH₂C(O)—; R⁴ is selected from the group consisting of carbonyl, —NH—, —S—, —CH₂—, and —O—; R⁵, R⁶, R⁷, and R⁸ are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, halo, hydroxy, trifluoromethyl, arylalkyl of 7-10 carbon atoms, aminoalkyl of 1-6 carbon atoms, hydroxyalkyl of 1-4 carbon atoms, alkoxy of 1-6 carbon atoms, carbalkoxy of 2-7 carbon atoms, cyano, amino, -C02H, or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from the group consisting of alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, and —CO₂H; R⁹ is hydrogen, alkyl of 1-6 carbon atoms, or aralkyl of 7-10 carbon atoms; b=0-10; d=0-10; e=0-2; x=0-1; y=0-1; z is from about 1 to about 120; and Carrier is immunogenic carrier material, detector carrier material, or a solid matrix, with the proviso that R¹ and R² are both not hydrogen; and further provided that when b is greater than 1, each of the CR⁵R⁶ groups can be the same or different, and when d is greater than 1, each of the CR⁷R⁸ groups can be the same or different; and still further provided that x is 0 if R¹ is hydrogen and y is 0 if R² is hydrogen, and if x and y are both 1, the Carrier moiety is the same in both cases.
 3. A rapamycin conjugate represented by formula III, or a salt thereof:

wherein R¹ and R² are each, independently, hydrogen or —(R³-L¹-R⁴)_(f)—(R¹⁰-L²-R¹¹)_(g)—; L¹ is —(CH₂)_(h)—CHR¹²—(CH₂)_(j)—; L² is —(CH₂)_(k)-D-(CH₂)_(m)-E-; D is —CH₂—, —S—S—, or

E is —CH₂— or —C—;

R³ and R¹⁰ are each, independently, selected from the group consisting of carbonyl, —S(O)—, —S(O)₂—, —P(O)₂—, —P(O)(CH₃)—, —C(S)—, and —CH₂C(O)—; R⁴ and R¹¹ are each, independently, selected from the group consisting of carbonyl, —NH—, —S—, —CH₂—, and —O—; R¹² is hydrogen, alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, —(CH₂)_(n)CO₂R¹³, —(CH₂)_(p)NR¹⁴R¹⁵, carbamylalkyl of 2-3 carbon atoms, aminoalkyl of 1-4 carbon atoms, hydroxyalkyl of 1-4 carbon atoms, guanylalkyl of 2-4 carbon atoms, mercaptoalkyl of 1-4 carbon atoms, alkylthioalkyl of 2-6 carbon atoms, indolylmethyl, hydroxyphenylmethyl, imidazoylmethyl, halo, trifluoromethyl, or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from the group consisting of alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, and —CO₂H; R¹⁴ and R¹⁵ are each, independently, hydrogen, alkyl of 1-6 carbon atoms, or arylalkyl of 7-10 carbon atoms; R¹³ is hydrogen, alkyl of 1-6 carbon atoms, arylalkyl of 7-10 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or phenyl which is optionally mono-, di-, or tri-substituted with a substituent selected from the group consisting of alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7 carbon atoms, trifluoromethyl, amino, and —CO₂H; f=0-3; g=0-1; h=0-10; j=0-10; k=0-10; m=0-10; n=0-6; p=0-6; x=0-1; y=0-1; z is from about 1 to about 120; and Carrier is immunogenic carrier material, detector carrier material, or a solid matrix, with the proviso that R¹ and R² are both not hydrogen; and further provided that f and g are both not 0 and when f is greater than 1, each of the —(R³-L¹-R⁴)— moieties can be the same or different; and still further provided that x is 0 if R¹ is hydrogen and y is 0 if R² is hydrogen, and if x and y are both 1, the Carrier moiety is the same in both cases.
 4. The rapamycin conjugate of claim 1, wherein said immunogenic carrier material is selected from the group consisting of keyhole limpet hemocyanin and ovalbumin.
 5. The rapamycin conjugate of claim 2, wherein said immunogenic carrier material is selected from the group consisting of keyhole limpet hemocyanin and ovalbumin.
 6. The rapamycin conjugate of claim 2, wherein said immunogenic carrier material is selected from the group consisting of keyhole limpet hemocyanin and ovalbumin.
 7. An immunoassay kit for measuring the blood level of a rapamycin comprising a conjugate of any one of claims 1, 2 or 3, wherein the carrier is a detector carrier material.
 8. The immunoassay kit of claim 1, wherein said detector carrier material is horseradish peroxidase.
 9. The rapamycin conjugate of claim 1, wherein in formula I, R¹ and R² are each —(R³-L-R⁴)_(a)—; R³ is carbonyl; L is a CH₂CH₂ linking group; R⁴ is a carbonyl, a is 1, x is 1, and y is
 0. 10. The conjugate of claim 3, wherein in formula III, R¹ is (R³-L¹-R⁴)_(f)—(R¹⁰-L²-R¹¹)_(g)—, R² is hydrogen, R³ is carbonyl; R⁴ is NH, and h is 0, R¹² is hydrogen, j is 0, and f is 1; R¹⁰ is carbonyl is 0, D is

m is 3, E is

and R¹¹ is NH, g is 1, x is 1 and y is 0, and Carrier is horseradish peroxidase.
 11. A conjugate represented by formula III

wherein R¹ is (R³-L¹-R⁴)_(f)—(R¹⁰-L²-R¹¹)_(g)—, R² is hydrogen, R³ is carbonyl, f is 1 and L¹ is (CH₂)_(h)—CHR¹²—(CH₂)_(j), h is 0, R¹² is hydrogen, j is 0; R⁴ is NH, R¹⁰ is carbonyl; L² is (CH₂)_(k)-D-(CH₂)_(m)-E, k is 1, D is

m is 0, E is carbonyl, R¹¹ is NH, g is 1, x is 1 and y is 0, and Carrier is horseradish peroxidase. 